**3. Targets of directed evolution technology**

*Current Topics in Biochemical Engineering*

[23, 24], and in vitro displays use a cell-free system. Cell-free systems contain components for protein synthesis such as ribosome, energy regeneration substrates, amino acids, and cofactors. Crude extract purified from wheat germ or insect cells [25, 26] and PURE system [27], a mixture of components separately purified from *Escherichia coli* cells, can be used as cell-free system. Protein can form normal

In vitro display is a protein which is connected to a DNA or RNA variant coding the protein, and various types of this display were proposed [7]. In RNA display, RNA variants combined with puromycin via linker molecules at 3′ terminal of mRNA are used. Puromycin stops the protein synthesis by combining to C-terminus of protein; the protein combined with the mRNA is produced. In ribosome display, RNA variants of which stop codon is removed are used. The ribosome cannot be demolished because of loss of the stop codon; the complex composed of mRNA, protein, and ribosome is produced. In DNA display, the biotin-labeled DNA variant which codes streptavidin gene at the terminal of the gene is used, and protein is synthesized in W/O emulsion. The protein tagged by streptavidin combines with biotin-labeled DNA. Liposome display, a single vesicle liposome containing the cell-free synthesis system, is used. Advantage of liposome display is that membrane

Target proteins must be screened from displays. In case of screening based on coupling constant, selection of protein is performed based on strength of binding with a target molecule immobilized on a plate or micro-beads. In the case of screening based on enzyme activity, the SIMPLEX method using microplate [28] can be used for the selection of DNA, RNA, and ribosome displays. Display using emulsion [29] and liposome display [30] is more useful for high-throughput screening because FACS or confocal fluorescence coincidence analysis (CFCA) [31] can be used. The advantage of in vitro display using a cell-free system is that construction

In vivo system using *E. coli* cells is also used for screening of proteins. The most general procedure is construction of a gene library of *E. coli* transformants: Plasmids containing DNA variants are transformed into *E. coli* cells. Phage display and display using virus-like molecules were also proposed. Phage display, which was developed to display V-region of antibody, can display the target protein at the surface of the phage by the fusion with coat protein of bacteriophage. Relatively large proteins can be displayed by the fusion with N-terminus of g3p protein of M13 phage or C-terminus of g10 protein of T7 phage [32]. Recently, several groups developed display using nucleocapsids as artificial virus [33, 34]. Non-viral cage of lumazine synthase (AaLS)

Selection method of target protein in in vivo displays is as follows: In case of the selection based on bonding strength, protein can be selected with phage display [32]. Biopanning is generally used for the selection from phage display: Phage displays connecting with immobilized molecules are collected and are transformed into *E. coli* cells. In the case of the selection based on enzyme activity, agar plate containing screening medium, which is the general method for screening of microorganisms, is used in the selection of *E. coli* transformant library. Besides this method, a method such as IAN-PCR method, which is based on amino acid sequence, is proposed. The procedure using *E. coli* library is not adapted to select from a huge size of library because the screening speed is very low, but this screening method is adapted to select from small-scale library such as site-mutated library

was spontaneously formed, and mRNA could be contained in the capsule.

combined with computer simulation or metagenome library.

tertiary structure by the addition of adequate chaperones.

protein can be displayed at surface of lipid bilayer.

of library and automation at microscale are easy.

*2.4.2 In vivo displays*

**4**

Directed evolution is a trend in twenty-first century, and interesting study subjects have been proposed [35]. One of the interesting study subjects is, of cause, development of super-proteins which are not present in the nature world and can catalyze novel reaction, and the study on Kemp elimination reaction is its pioneer. Natural enzyme catalyzing the Kemp elimination reaction had not been discovered. Rӧthlisberger et al. produced the eight novel enzymes catalyzing Kemp elimination reaction by using computational design and site mutagenesis [36]. Similarly, Hilvert et al. conducted the site-directed mutagenesis and screened by using dropletbased microfluidic screening platform. The obtained artificial aldolases showed high activity although the original protein showed few activities [37]. Tryptophan synthetase and cytochrome P450 variants obtained by directed evolution could catalyze novel reactions [38]. In addition to directed evolution, metagenomics is a powerful tool to discover such unknown enzymes [39, 40]. For instance, more than unknown 300 nitrilases were discovered from the metagenome library, although nitrilase discovered during many years of study was only less than 20 [41].

Directed evolution technology can be also applied to improvement of metabolic pathways [42]. For example, 1-propanol is expected as a biofuel, but adequate producing strains have not been discovered. Thus, Atsumi et al. produced *E. coli* recombinant by expressing a series of genes for 1-propanol production, and improvement of citramalate synthase gene was conducted by using error-prone PCR and DNA shuffling. The productivities of 1-propanol and 1-butanol in the improved strain were enhanced [43]. Arnold et al. improved alcohol dehydrogenase and ketol-acid reducto-isomerase to enhance the productivity of 1-propanol by directed evolution. The obtained enzymes could use NADH instead of NADPH as a coenzyme [44]. Otherwise, directed evolution technology is applied to regulatory genes for improvement of metabolic pathway. Promoter [45], operon connection [46], and enhancer sequences [47] were improved by the method.

The other interesting study subject is to understand the evolution of life. One approach to the subject is to know which genome length is required for a microorganism. Gibson et al. constructed artificial genome DNA which only includes chemically synthesized and introduced the genome into microorganisms in which the genome was previously removed [48]. Another research group tried to construct minimal bacterial genome; the constructed genome JCVI-syn3.0 was only 531 kb (473 genes) [49], and the obtained microorganism could grow and showed several characteristics of the microorganism.

The other approach to understand evolution of life is to generate artificial cells using a cell-free system. Szostak et al. randomly synthesized RNAs (1015 of 90 mer) and created a ribozyme having sufficient RNA ligase activity using error-prone PCR with only 10 rounds of repeats [50]. Noireaux et al. constructed cell-sized synthetic vesicle (artificial cells) containing components for translation and transduction [51]. Yomo et al. also produced novel artificial cells, which can progress artificial evolution of RNAs by themselves [52]. Induction of unnatural compounds into cells is another approach. Unnatural basic pairs and more than 100 unnatural amino acids were synthesized, and they were site-specifically introduced into proteins [53]. Accordingly, evolutional RNA engineering may impart validity to the hypothesis of RNA word in the future.
